KINASES AND CONTROL OF CELL-TYPE SPECIALIZATION

丝裂原活化蛋白激酶（MAPK）级联由三种顺序作用的蛋白激酶组成。相关级联反应在细胞中重复，以介导对细胞外刺激的不同反应。尽管这种情况存在广泛的串扰的可能性，但通常观察到给定途径的MAPK仅由适当的刺激激活。这种特异性似乎得以维持，因为给定级联的酶在稳定的复合物中彼此缔合。这种模块化组织对信号放大，调节和整合的影响对于理解控制正常增殖，发育和炎症反应的事件至关重要。虽然脊椎动物通路的模块化组织变得越来越明显，但酵母中的类似通路是这些级联的架构和动力学的范例。我们将利用已知的酵母交配压力和细胞完整性级联来了解模块化组织对信号传输机制和通路间通信的影响。我们的目标是：[1]定义支架协会在信号传导中的调节和功能。分离与支架-Ste 5结合有缺陷的MAPK激酶Ste 7突变体，以鉴定结合区域并了解磷酸化如何调节Ste 7-Ste 5缔合。这些突变体也将被用作工具，以了解Ste 7-Ste 5缔合在体内信号传导和体外磷酸转移反应中的作用。[2]鉴定控制信号传导组件的开启和关闭状态之间转换的蛋白质。两个混合筛选和生化方法将被用来隔离监管机构，促进过渡从非生产性的生产性相互作用的Ste 7与其他组件的交配途径模块。[3]调查是否共享组件的分布调节通路活动。 将分离MAPK激酶激酶Ste 11的结合缺陷突变体，其区分与交配模块中的Ste 5和应激模块中的MAPK激酶Pbs 2的结合，并用于了解其与一个模块的相互作用是否限制了另一个模块中的信号传导。Pbs 2-Ste 11共定位将在体内使用共聚焦荧光显微镜检查信号传导期间。{4}定义一个刺激激活两个MAPK通路的机制。信息素激活配对MAPK级联，其输出然后刺激细胞完整性MAPK级联。遗传学方法将被用来确定“第二信使”协调这两个途径的活动。

Mitogen activated protein kinase (MAPK) cascades are comprised of three sequentially acting protein kinases. Related cascades are reiterated in the cell to mediate distinct responses to a host of extracellular stimuli. Despite the potential this situation presents for extensive cross talk, it is generally observed that the MAPKs of a given pathway are activated only by the appropriate stimuli. This specificity appears to be maintained because the enzymes of a given cascade are associated with each other in stable complexes. The ramifications that this modular organization has on signal amplification, regulation and integration is central to understanding events that control normal proliferation, development and inflammatory responses. While a modular organization of vertebrate pathways is becoming apparent, analogous pathways in yeast are the paradigms for the architecture and dynamics of these cascades. We will exploit what is known about the yeast mating stress and cell- integrity cascades to learn what consequences a modular organization has on the mechanics of signal transmission and on inter-pathway communication. Our aims are to: [1] Define the regulation and function of scaffold associations in signaling. Mutants of the MAPK kinase Ste7 that are defective for binding to the scaffold-Ste5 will be isolated to identity the binding region and learn how phosphorylation regulates the Ste7-Ste5 association. The mutants also will be used as tools to learn what role the Ste7-Ste5 association has on signaling in vivo and on phosphotransfer reactions in vitro. [2] IdentifY protein(s) controlling transitions between on and off states of signaling assemblies. Two hybrid screens and a biochemical approach will be used to isolate regulators that facilitate a transition from a non-productive to productive interaction of Ste7 with other components of the mating pathway module. [3] Investigate whether distribution of shared components regulates pathway activities. Binding defective mutants of the MAPK kinase kinase Ste11 that discriminate between binding to Ste5 in the mating module and the MAPK kinase Pbs2 in the stress module will be isolated and used to learn if its interaction with one module limits signaling in the other. Pbs2-Ste11 co-localization will be examined in vivo during signalling using confocal fluorescence microscopy. {4} Define the mechanism by which one stimulus activates two MAPK pathways. Pheromone activates the mating MAPK cascade whose output then stimulates the cell-integrity MAPK cascade. Genetic methods will be used to identity the "second messengers" coordinating the of activities of these two pathways.